Environmental stress due to salinity is one of the most serious factors limiting the productivity of agricultural crops, which are predominantly sensitive to the presence of high concentrations of salts in the soil. Large terrestrial areas of the world are affected by levels of salt inimical to plant growth. It is estimated that 35-45% of the 279 million hectares of land under irrigation is presently affected by salinity. This is exclusive of the regions classified as arid and desert lands, (which comprise 25% of the total land of our planet). Salinity has been an important factor in human history and in the life spans of agricultural systems. Salt impinging on agricultural soils has created instability and has frequently destroyed ancient and recent agrarian societies. The Sumerian culture faded as a power in the ancient world due to salt accumulation in the valleys of the Euphrates and Tigris rivers. Large areas of the Indian subcontinent have been rendered unproductive through salt accumulation and poor irrigation practices. In this century, other areas, including vast regions of Australia, Europe, southwest USA, the Canadian prairies and others have seen considerable declines in crop productivity.
Although there is engineering technology available to combat this problem, through drainage and supply of high quality water, these measures are extremely costly. In most of the cases, due to the increased need for extensive agriculture, neither improved irrigation efficiency nor the installation of drainage systems is applicable. Moreover, in the arid and semi-arid regions of the world water evaporation exceeds precipitation. These soils are inherently high in salt and require vast amounts of irrigation to become productive. Since irrigation water contains dissolved salts and minerals, an application of water is also an application of salt that compounds the salinity problem.
Increasing emphasis is being given to modify plants to fit the restrictive growing conditions imposed by salinity. If economically important crops could be manipulated and made salt resistant, this land could be farmed resulting in greater sales of seed and greater yield of useful crops. Conventional breeding for salt tolerance has been attempted for a long time. These breeding practices have been based mainly on the following strategies: a) the use of wide crosses between crop plants and their more salt-tolerant wild relatives (Rush and Epstein, J. Amer. Hort. Sci., 106:699-704 (1981)), b) screening and selecting for variation within a particular phenotype (Norlyn, in Genetic Engineering of Osmoregulation, pp. 293-309 (1980)), c) designing new phenotypes through recurrent selection (Tal, Plant & Soil, 89:199-226 (1985)). The lack of success in generating tolerant varieties (given the low number of varieties released and their limited salt tolerance) (Flowers and Yeo, Aust. J, Plant. Physiol., 22:875-884 (1995)) would suggest that conventional breeding practices are not enough and that in order to succeed a breeding program should include the engineering of transgenic crops (Bonhert and Jensen, Aust. J. Plant. Physiol., 23:661-667 (1996)).
Several biochemical pathways associated with stress tolerance have been characterized in different plants and a few of the genes involved in these processes have been identified and in some cases the possible role of proteins has been investigated in transgenic/overexpression experiments. Several compatible solutes have been proposed to play a role in osmoregulation under stress. Such compatible solutes, including carbohydrates (Tarcynski et al., Science, 259:508-510 (1995)), amino acids (Kishor et al., Plant Physiol., 108:1387-1394 (1995)) and quaternary N-compounds (Ishtani et al., Plant Mol. Biol., 27:307-317 (1995)) have been shown to increase osmoregulation under stress. Also, proteins that are normally expressed during seed maturation (LEAs, Late Embriogenesis Abundant proteins) have been suggested to play a role in water retention and in the protection of other proteins during stress. The overexpression of LEA in rice provided a moderate benefit to the plants during water stress (Xu et al., Plant Physiol., 110:249-257 (1996), and Wu and Ho, WO 97/13843).
A single gene (sod2) coding for a Na+/H+ antiport has been shown to confer sodium tolerance in fission yeast (Jia et al., EMBO J., 11:1631-1640 (1992) and Young and Zheng, WO 91/06651), although the role of this plasma membrane-bound protein appears to be only limited to yeast. One of the main disadvantages of using this gene for transformation of plants is associated with the typical problems encountered in heterologous gene expression, i.e. incorrect folding of the gene product, targeting of the protein to the target membrane and regulation of the protein function.
Na+/H+ antiporters with vacuolar antiport activity have been identified in red beet storage tissue and a variety of halophytic and salt-tolerant glycophtic plant species (Barkla and Pantoja, Ann. Rev. Plant. Physiol. 47:159 (1996), and Blumwald and Gelli, Adv. Bot. Res. 25:401 (1997)). More recently, a gene encoding a vacuolar Na+/H+ antiporter from Arabadopsis thaliana, designated AtNHX1, has been isolated (Blumwald et al., WO 99/47679). Overexpression of this gene in Arabadopsis, tomato, and canola has been shown to enhance salt tolerance in transgenic plants (Apse et al., Science, 285:1256-1258 (1999), Zhang and Blumwald, Nat. Biotechnol, 19:765-768 (2001), and Zhang et al., PNAS USA, 98:12832-12836 (2001)).